The primary goal of this grant proposal is to demonstrate that self-association of apolipoprotein A-I (apoA-I) is a functional feature of the protein and that alteration of its native state of self-association is involved in mechanisms leading to disease. Self-association is an inherent property of the lipid-free forms of several exchangeable apolipoproteins, including apoA-I, the main protein component of HDL and an established antiatherogenic factor. Monomeric lipid-free apoA-I is believed to be the biologically active species. But abnormal conditions, such as specific mutations or oxidation, produce an altered state of self-association that may contribute to apoA-I dysfunction. Although the functional role of self-association in some apolipoproteins has been established, the influence of self-association on apoA-I function has not been studied before because of technical limitations that are addressed and overcome in this project. Replacement of apoA-I's Trps with Phes (W-apoA-I) leads to unusually large and stable self-associated species. At least four self-associated species of W-apoA-I can be isolated and will be used here as a model of self-association to analyze its role in determining apoA-I's structure, function, and susceptibility to mechanisms leading to dysfunction. The three overlapping areas to be investigated in this project are: 1. Define the effects of self-association at all levels of apoA-I structure, from secondary to quaternary. The nature of the inter-molecular interactions that are involved in apoA-I self-association will be established and the structural details underlying the loss of lipid-binding efficiency for increasing degrees of self- association will be determined. This structural knowledge will help to understand the mechanisms whereby alteration of the protein self-association state affects its biological function. 2. Characterize how the self-association state o lipid-free apoA-I affects its function as recipient of lipids released from cells in the biogenesisof HDL. The efficiency of different apoA-I self-associated species in activating lipid release mediated by different cell membrane transporters will be determined. Including ABCA1, which is the primary mechanism underlying the anti-atherogenic function of apoA-I. a. Demonstrate that the self-association state of lipid-free apoA-I modulate the protein susceptibility to mechanisms leading to dysfunction. The vulnerability of different self-associated species to reactions that are implicated in the pathogenesis of atherosclerosis and diabetes will be tested. The possible role of self-association in protecting apoA-I from conditions which promote amyloid fibril formation, a contributing mechanism to atherosclerosis progression, will be also evaluated. These studies are highly significant for public health because determining a new functional aspect of apoA- I, which is one of the most important known anti-atherogenic factors, bears potential for the formulation of new therapies and the development of new biomarkers for the evaluation of cardiovascular disease risk.